p16INK4a (p16) traps the cell at the restriction point of the cell cycle by binding to cyclin-dependent kinase 4/6 thus preventing the phosphorylation of the retinoblastoma protein (pRB). As p16 accumulates the cell stops dividing and becomes senescent. This study investigates the modulation of p16 function by the putative membrane protein ABCB5 and a group of five putative oncogenic microRNAs (oncomiRs). ABCB5 is a poorly characterised member of the B-subfamily of human ATP Binding Cassette transporters. ABCB5 is reportedly transcribed into four transcripts, one of which could potentially encode a full-length transporter (ABCB5fl) whilst a second could encode a half-transporter (ABCB5β). The other two transcripts (ABCB5α and ABCB5γ) could only encode short polypeptides. Exogenous expression of ABCB5fl and ABCB5β was achieved in HEK293T cells, but the recombinant protein expressed poorly and localised to the endoplasmic reticulum. Point mutations introduced into the ATP catalytic domain failed to improve expression levels suggesting that protein function was not deleterious to the cell. Exogenous expression in HEK293T cells also allowed commercial antibodies purportedly raised against ABCB5 isoforms to be tested. Several were found not to recognise ABCB5 necessitating re-interpretation of published data. However, one antibody recognised both ABCB5fl and ABCB5β, and was subsequently used to evaluate protein expression levels in other cell types.siRNA knockdown of ABCB5 in human mammary epithelial cells (HMECs) caused a concomitant reduction in p16 expression and an increase in cellular proliferation. Differential siRNAs and RT-qPCR analyses demonstrated ABCB5β to be the relevant transcript with respect to the reduction in p16 expression; however, no native ABCB5β protein was detected in HMECs. Together these data lead to the hypothesis that the ABCB5β transcript may act as a long noncoding RNA to regulate p16. Exogenous expression of each of five distinct putative oncomiRs in HMECs was found to increase cellular proliferation and, surprisingly, increase p16 expression. These results mirror a phenotype commonly observed in p16-positive basal-like breast cancer (BLBC), an aggressive form of breast cancer with poor prognosis and few treatment options. Bioinformatic analysis of the predicted target genes for these oncomiRs identified multiple transcriptional regulators of pRB. These predictions, together with the work performed in a cellular model of p16-positive BLBC, suggest that the oncomiRs may cause unrestricted cell proliferation by indirectly reducing transcription of the pRB gene, RB1. In the absence of pRB, p16 expression is induced via a previously reported oncogeneinduced senescence-like positive feedback loop. These data, and previously published observations, suggest that a similar mechanism may explain the basis of p16-positive BLBC.

Background: Very recently a gene marker panel that allows the mutational analysis of APC, CTNNB1, B-RAF and K-RAS was conceived. The aim of the present study was to use the 4-gene marker panel covering the Wnt and Ras-Raf-MEK-MAPK signalling pathways to determine the percentage of sporadic colorectal carcinomas (CRC) carrying at least one of the four above-mentioned genes in a mutated form alone and/or in combination with microsatellite instability (MSI) and to compare the sensitivity of the gene marker panel used in this study with that of gene marker panels previously reported in the scientific literature. Methods: CTNNB1 and B-RAF were screened by PCR-single-strand conformation polymorphism analysis and K-RAS gene mutations by restriction fragment length polymorphism analysis. For the mutational analysis of the APC gene mutation cluster region (codons 1243–1567) direct DNA sequencing was performed. The U.S. National Cancer Institute microsatellite panel (BAT25, BAT26, D2S123, D5S346 and D17S250) was used for MSI analysis. Results: It could be shown that about 80% of early stage CRC (UICC stages I and II) and over 90% of CRC in the UICC stage IV carried at least one mutated gene and/or showed MSI. No significant increase in the gene mutation frequencies could be determined when comparing tumours in the UICC stage I with those in UICC stage IV. Conclusions: When compared with previously published gene marker panels the 4-gene marker panel used in the present study shows an excellent performance, allowing to detect genetic alterations in 80–90% of human sporadic CRC samples analyzed.

Familial Pancreatic Cancer (FPC) is a cancer syndrome characterized by clustering of pancreatic cancer in families, but most FPC cases do not have a known genetic etiology. Understanding genetic predisposition to pancreatic cancer is important for improving screening as well as treatment. The central aim of this thesis is to identify candidate susceptibility genes for FPC, and I used three approaches of increasing resolution. First, based on a candidate-gene approach, I hypothesized that BRCA1 is inactivated by loss-of-heterozygosity in pancreatic adenocarcinoma of germline mutation carriers. I demonstrated that 5/7 pancreatic tumors from BRCA1-mutation carriers show LOH, compared to only 1/9 sporadic tumors, suggesting that BRCA1 inactivation is involved in tumorigenesis in germline mutation carriers. Second, I hypothesized that the germline genomes of FPC subjects differ in copy-number profile from healthy genomes, and that regions affected by rare deletions or duplications in FPC subjects overlap candidate tumor-suppressors or oncogenes. I found no significant difference in the global copy-number profile of FPC and control genomes, but I identified 93 copy-number variable genomic regions unique to FPC subjects, overlapping 88 genes of which several have functional roles in cancer development. I investigated one duplication to sequence the breakpoints, but I found that this duplication did not segregate with disease in the affected family. Third, I hypothesized that in a family with multiple pancreatic cancer patients, genes containing rare variants shared by the affected members constitute susceptibility genes. Using next-generation sequencing to capture most bases in coding regions of the genome, I interrogated the germline exome of three relatives who died of pancreatic cancer and a relative who is healthy at advanced age. I identified a short-list of nine candidate genes with unreported mutations shared by the three affected relatives and absent in the unaffected relative, of which a few had functional relevance to tumorigenesis. I performed Sanger sequencing to screen an unrelated cohort of approximately 70 FPC patients for mutations in the top two candidate genes, but I found no additional rare variants in those genes. In conclusion, I present a list of candidate FPC susceptibility genes for further validation and investigation in future studies.

Familial Pancreatic Cancer (FPC) is a cancer syndrome characterized by clustering of pancreatic cancer in families, but most FPC cases do not have a known genetic etiology. Understanding genetic predisposition to pancreatic cancer is important for improving screening as well as treatment. The central aim of this thesis is to identify candidate susceptibility genes for FPC, and I used three approaches of increasing resolution. First, based on a candidate-gene approach, I hypothesized that BRCA1 is inactivated by loss-of-heterozygosity in pancreatic adenocarcinoma of germline mutation carriers. I demonstrated that 5/7 pancreatic tumors from BRCA1-mutation carriers show LOH, compared to only 1/9 sporadic tumors, suggesting that BRCA1 inactivation is involved in tumorigenesis in germline mutation carriers. Second, I hypothesized that the germline genomes of FPC subjects differ in copy-number profile from healthy genomes, and that regions affected by rare deletions or duplications in FPC subjects overlap candidate tumor-suppressors or oncogenes. I found no significant difference in the global copy-number profile of FPC and control genomes, but I identified 93 copy-number variable genomic regions unique to FPC subjects, overlapping 88 genes of which several have functional roles in cancer development. I investigated one duplication to sequence the breakpoints, but I found that this duplication did not segregate with disease in the affected family. Third, I hypothesized that in a family with multiple pancreatic cancer patients, genes containing rare variants shared by the affected members constitute susceptibility genes. Using next-generation sequencing to capture most bases in coding regions of the genome, I interrogated the germline exome of three relatives who died of pancreatic cancer and a relative who is healthy at advanced age. I identified a short-list of nine candidate genes with unreported mutations shared by the three affected relatives and absent in the unaffected relative, of which a few had functional relevance to tumorigenesis. I performed Sanger sequencing to screen an unrelated cohort of approximately 70 FPC patients for mutations in the top two candidate genes, but I found no additional rare variants in those genes. In conclusion, I present a list of candidate FPC susceptibility genes for further validation and investigation in future studies.

Although many group I (GI) introns can self-splice in vitro, their splicing is promoted by proteins in vivo. Only a few splicing factors that specifically promote GI intron splicing have been identified, however, none are from chloroplasts, which is the subject of this study. In previous work from our lab, a strategy was developed to identify splicing factors for chloroplast GI introns of Chlamydomonas by using suppressor genetics. A mutant with reduced splicing of the chloroplast 23S rRNA intron (Cr.LSU) was generated. Then, 3 nuclear suppressors (7120, 71N1 and 7151) with substantially restored splicing of Cr.LSU were isolated and partially characterized. However, the suppressor gene(s) were not identified. In this study, I have used genetic mapping to make a renewed attempt to isolate these genes. Using polymorphisms between the 137C strain that was used for suppressor isolation, and a new strain of C.reinhardtii (S1D2), the nuclear suppressor mutations in 7120 and 71N1 were mapped to a region on chromosome III that is essentially devoid of recombination. Based on the recombination maps, the suppressor gene in 7120 is located within a ~418-kb region from bp 2,473,064 to 2,891,232, whereas the suppressor in 71N1 is likely located within a ~236-kb subregion from bp 2,473,064 to 2,709,377. It is possible that these mutations are in the same gene; however, the maps could not be refined further due to the lack of recombination in this 418-kb region. I also attempted to compare the genomic sequence of the 7120 suppressor, which was obtained by next-generation sequencing, with the Chlamydomonas reference genome (JGI, v.4). Next-generation sequencing of 7120 revealed the existence of abundant repetitive sequences and transposable elements clustered in a ~40-kb subregion of the recombinationally suppressed 418-kb region on chromosome III. I suggest that the high frequency of repetitive sequences and transposable elements in this region may be the reason for the suppressed recombination. Searching for candidate genes in the mapped region led me to examine a novel protein that was predicted to have a putative chloroplast transit-peptide, and an RNA binding domain. Further bioinformatic analysis revealed a single rhodanese domain with an active-site cysteine. The protein was expressed in E.coli as the full-length and predicted mature forms, plus a small His-tag. The purified mature protein had rhodanese catalytic activity, based on the fact that it was able to transfer sulfur from thiosulfate to cyanide. Also, western blot analysis with a polyclonal antibody produced in rabbits showed that the cellular protein migrated on SDS gels close to the mature protein and faster than the full-length protein, indicative of an organelle-targeted protein. The antibody also showed that the cellular protein co-fractionated with chloroplasts. To gain insight into its in vivo function, the gene was knocked down using the tandem RNAi system (Rohr et al., 2004), which produced strains (5) with reductions of 31% to 76% in the mRNA level, and ~30% to ~60% in the protein level. These strains were sensitive to bright light, and had reduced rates of growth under all conditions, which are characteristics of chloroplast translation mutants. Thus, chloroplast protein synthesis was examined by radioisotope pulse-labeling in the presence of cycloheximide, which showed that the RNAi strains were broadly and negatively affected, and RT-PCR and northern blot revealed only normal chloroplast mRNA levels. These data have identified a new rhodanese-family enzyme that is required for chloroplast translation, which I have designated “CRLT”, for chloroplast rhodanese-like translation. / text

Orientador: Maria Inês de Moura Campos Pardini / Banca: Adriana Camargo Ferrassi / Banca: José Roberto Fígaro Caldeira / Resumo: O câncer de mama é o tipo de câncer mais comum entre as mulheres, correspondendo a 22% de todos os casos. Foram estimados mais de 1.050.000 casos novos de câncer de mama em todo o mundo no ano 2000. Mecanismos epigenéticos como a ativação e desativação de genes por meio da hipometilação e hipermetilação, respectivamente, da região promotora dos genes estão associados ao surgimento de diversos tipos de cânceres. Embora os fatores que resultam na metilação aberrante ainda não sejam bem conhecidos, a deficiência de folato têm sido associada a esse mecanismo no desenvolvimento de cânceres como de colo uterino, pulmão, mama, cólon e cérebro. Neste trabalho, foi avaliado se os polimorfismos em genes de enzimas chaves no metabolismo do folato como a timidilato sintase (TS) e metilenotetrahidrofolato redutase (MTHFR) influenciam o padrão de metilação da região promotora dos genes CDH1, BRCA1 e hMLH1 em amostras de câncer de mama. A metilação foi avaliada através da MS-PCR e os polimorfismos por PCR e PCR-RFLP. Não houve diferença estatisticamente significante (p<0.05, teste exato de Fisher) do padrão de metilação dos genes quando comparado com estádio, grau histológico, idade e freqüência dos polimorfismos. Os polimorfismos 5'UTR TS, C677T e A1298C MTHFR não influenciam no padrão de metilação da região promotora dos genes CDH1, BRCA1 e hMLH1 em amostras de câncer de mama / Abstract: The breast cancer is the most frequent cancer in women equivalent to 22% of all cases. It were estimated more than 1.050.000 new cases of breast cancer in all the world in the year 2000. Epigenetic mechanisms like ativation and desativation of genes by hypomethylation and hipermethylation, respectivelly, of genes promoter regions are associated to the appearance of several types of cancer. Although the factors that result in aberrant methylation are not well known, the lack of folate has been associated to this mechanism in the development of cancers like cervical uterine, lungs, breast, colon and brain. In this research it was evaluated if the polymorphisms in genes of folate metabolism enzymes like the thymidilate syntase (TS) and methylenetetrahydrofolate reductase (MTHFR) influence the methylation pattern of the promoter of genes CDH1, BRCA1 and hMLH1 in samples the tissue of breast cancer. The methylation was evaluated through the MS-PCR and the polymorphisms through PCR and PCR-RFLP. We observed no statistically significant associations (p<0.05, exact test of Fisher) of the patterns of methylation of genes when compared to stage, histologic grade, age and frequency of polymorphisms. The polymorphisms 5'UTR TS, C677T AND A1298C MTHFR did not influence in the pattern of methylation of the promoter region of genes CDH1, BRCA1 e hMLH1 in samples the tissue of breast cancer / Mestre

Formação de tumores deve-se a combinações de fatores. A via de p53 tem um papel fundamental no controle de proliferação e apoptose. O interferon-beta (IFNb) é importante na modulação da resposta imunológica, no efeito antitumoral e no impacto apoptótico em células tumorais. Segundo a literatura, IFNb ativa a transcrição de p53 e componentes do sistema IFN efetuam sua função pela via p53/p14arf. Neste projeto, foi construída uma série de ferramentas para explorar interações entre p53 e IFNb. A primeira ferramenta, uma linhagem celular derivada de B16 com expressão de p53 reduzida por miRNA. Também construímos vetores plasmidiais e adenovirais portadores dos cDNAs para eGFP, Luciferase, p53 ou IFNb. Os vetores são utilizados para introduzir estes fatores, sozinho ou combinados, na célula alvo. Mesmo confirmando a atividade de p53 ou IFNb sozinho, não foi observado um efeito aditivo destes fatores em conjunto com este tipo de ensaio. Futuros estudos das possíveis interações entre as vias de p53 e IFNb terão o benefício das ferramentas construídas neste projeto. / Formation of tumors it must to combinations of factors. The p53 pathway has an essential role in proliferation control and apoptosis. The interferon-beta (IFNb) is important in modulation of the immunologic response, in the antitumoral effect and in the apoptotic impact in tumor cells. According to literature, IFNb activate the p53 transcription and components of IFN system effect its function to p53/p14arf pathway. In this project, a series of tools was constructed to explore interactions between p53 and IFNb. The first tool, a cellular lineage derivative of B16 with expression of p53 reduced by miRNA. We also construct plasmidial and adenoviral vectors carriers of cDNAs for eGFP, Luciferase, p53 or IFNb. The vectors are used to introduce these factors, alone or agreed, in the target cell. Even confirming the activity of p53 or IFNb alone, an additive effect of these factors combined was not observed with this type of assay. Future studies of the possible interactions between p53 and IFNb pathways will have the benefit of the tools constructed in this project.

abstract: Cancer is a disease which can affect all animals across the tree of life. Certain species have undergone natural selection to reduce or prevent cancer. Mechanisms to block cancer may include, among others, a species possessing additional paralogues of tumor suppressor genes, or decreasing the number of oncogenes within their genome. To understand cancer prevention patterns across species, I developed a bioinformatic pipeline to identify copies of 545 known tumor suppressor genes and oncogenes across 63 species of mammals. I used phylogenetic regressions to test for associations between cancer gene copy numbers and a species’ life history. I found a significant association between cancer gene copies and species’ longevity quotient. Additional paralogues of tumor suppressor genes and oncogenes is not solely dependent on body size, but rather the balance between body size and longevity. Additionally, there is a significance association between life history traits and genes that are both germline and somatic tumor suppressor genes. The bioinformatic pipeline identified large tumor suppressor gene and oncogene copy numbers in the naked mole rat (Heterocephalus glaber), armadillo (Dasypus novemcinctus), and the two-fingered sloth (Choloepus hoffmanni). These results suggest that increased paralogues of tumor suppressor genes and oncogenes are these species’ modes of cancer resistance. / Dissertation/Thesis / Pipeline results for cancer genes / Phylogenetic regressions with correction tests / Pipeline results for housekeeping genes / Masters Thesis Biology 2019

The human genome is a dynamic structure, recently recognized to present with significant large-scale structural variation. DNA-copy number changes represent one common type of such variation and is found both between individuals and within the somatic cells of the same individual, especially in disease states like cancer.  Apart from DNA-rearrangements, epigenomic changes are increasingly acknowledged as important events in the maintenance of genomic integrity. In this thesis, different array-based methods have been applied for global genomic and epigenomic profiling of both normal and cancer cells. In paper I, a genomic microarray was established and used to determine DNA-copy number variants (CNVs) in a cohort of 76 healthy individuals from three ethnic populations. We identified 315 CNV regions that in total encompassed ~3,5% of the genome. In paper II, the array was utilized to discover CNVs within several differentiated tissues from the same subject. Six variants were identified providing evidence for somatic mosaicism. In paper III and IV we studied pheochromocytomas and paragangliomas, rare endocrine tumours that most often present as benign and sporadic with unclear genetic/epigenetic cause. Genome-wide DNA-copy number analysis of 53 benign and malignant samples in paper III revealed numerous common and novel chromosomal regions of losses and gains. High frequencies of relatively small overlapping regions of deletions were detected on chromosome 1p arm, encompassing several candidate tumour suppressor genes. In paper IV, an epigenomic map for two histone modifications associated with silent (H3K27me3) or active (H3K4me3) gene transcription, was generated for one malignant pheochromocytoma. Integrated analysis of global histone methylation, copy number alterations and gene expression data aided in the identification of candidate tumour genes. In conclusion, the performed studies have contributed to gain knowledge of CNVs in healthy individuals, and identified regions and genes which are likely associated with the development and progression of pheochromocytoma/paraganglioma.

Ce travail de recherche montre que les différentes sources de polyphénols (polyphénols de vin rouge, jus d'aronia melanocarpa, jus de cassis) ont de puissants effets chemothérapeutiques et chemopréventifs sur différentes lignées de culture cellulaires, mais également in vivo sur un modèle de tumorigenèse. Ces polyphénols inhibent la prolifération des cellules cancéreuses (leucémie lymphoblastique aigüe, cellules souches) en induisant un arrêt du cycle cellulaire et l'apoptose. Les effets anti-cancéreux sont dépendants de l' induction du stress oxydatif mettant en jeu les anions superoxydes et le peroxyde d·hydrogène qui à son tour, activent les voies de signalisation conduisant à une surexpression des gènes suppresseurs de tumeurs comme p73 et p53 et ainsi que caspase 3. Celle étude montre également que les polyphénols contrôlent la prolifération des cellules cancéreuses au niveau épigénétique en diminuant l'expression d'UHRF1 (un intégrateur épigénétique de prolifération). Cependant l'effet anticancéreux de ces polyphénols est sélectif et agit sur les cellules cancéreuses et non sur les cellules normales. Le fractionnement de ces sources riches en polyphénols et les études menées en utilisant des composés purs montrent que les effets anticancéreux sont attribués à plusieurs composés différents. Cette étude montre l' identification de cyanidine-3-glucoside et de cyanidine-3-rutinoside comme source de composés anticancéreux actifs. / This research work shows that different sources of polyphenols (RWPs, AMJ and blackcurrant) have strong chemotherapeutic and chemopreventive effects on several cancer cells lines (acute lymphoblastic leukemia and cancer stem cells) and also in vivo in a model of tumorigenesis in mouse. These polyphenols inhibit the proliferation of various cancer cells by inducing cell cycle arrest and apoptosis. The anti-cancer effect is dependent on the induction of oxidative stress involving superoxide anions and hydrogen peroxide which, in turn, activate the signaling pathways leading to the re-expression of tumor suppressor genes such as p73 and p53 and executor of apoptosis such as caspase 3. This study also shows that polyphenols control the proliferation of cancer cells at epigenetic level by decreasing the expression of UHRF1 (an epigenetic integrator of proliferation). Moreover, the anticancer effect of these polyphenols is selective towards cancer cells and not in normal cells. Fractionation of these rich sources of polyphenols and studies on the commercially available pure products shows that anti-cancer effects of these polyphenols involve several different compounds. This study leads to the identification of cyaniding-3-O-glucoside and cyaniding-3-O-rutinoside as active anticancer compounds.